Abstract/Summary In this competitive renewal application of the Program Program grant >30 yrs in existence, diverse approaches are used to address the singular theme of gene regulation in developing red blood cells. The Project Leaders are united in their commitment to the developing red cell as an experimental system in which to make fundamental biological discoveries that will inform our understanding of cell differentiation and directly impact our capacity to treat disorders of the red cell, including congenital and acquired anemias. The Program encompasses the most relevant vertebrate systems (human, mouse, and zebrafish) and employs contemporary genetic approaches, including gene modification using CRISPR/cas9 and innovative genetic screens. Deep, longstanding, and intensive interactions and collaborations among the Project Leaders ensures synergy in the accomplishment of the proposed goals of the research. Project 1 (S.H. Orkin, Project Leader) builds on prior groundbreaking work on the role of BCL11A in HbF repression and centers on the interaction of BCL11A and other transcription factors with the nuclear matrix, the turnover of BCL11A protein and the search for small molecules that destabilize the protein in erythroid cells, and cis-regulatory sites of BCL11A-binding in the human ?-globin cluster. In Project 2, H. Lodish (Project Leader) and his colleagues focus on the pathways by which self-renewal of red cell progenitors is controlled. Project 3 (L. Zon, Project Leader) addresses the role of transcription elongation in red cell gene expression and its intersections with signaling pathways. In Project 4 (D.E. Bauer, Project Leader), erythroid cell super-enhancer elements will be analyzed functionally with innovative CRISPR/cas9 saturating mutagenesis, an approach recently pioneered in the Program. In Project 5 (B. Paw, Project Leader), new genes/proteins involved in iron and heme metabolism will be characterized for their physiological roles in mouse/human/zebrafish systems. Each of the projects in this Program grant addresses critical aspects of the expression program and function of erythroid cells. These studies are founded on the premise that discoveries emerging from the proposed projects will provide new opportunities for the design of novel approaches to the understanding and management of red blood cell disorders, including the hemoglobinopathies (sickle cell disease and ?-thalassemia), other congenital anemias, and acquired conditions.